Method of inhibition of cellular and molecular level biological interactions utilizing non-peptidic surrogates of the Arg-Gly-Asp sequence

ABSTRACT

A method of inhibition of cellular and molecular level biological interactions which are dependent on RGD interactions is disclosed. The method comprises administering a compound of the formula I 
     
         H.sub.2 N--C(═NH)--NH--CH.sub.2 --A--CH.sub.2 --CO.sub.2 H (I) 
    
     and pharmaceutically acceptable salts thereof, wherein A is a chain of 9 atoms selected from the group consisting of: ##STR1## wherein each of x, n and m is at least 1; in chains (i) and (ii) n is at most 6; in chains (iii) and (iv) the sum of x+n+m is 5; and the sum of n+m is 4 in chain (v) and 3 in chain (vi).

This application is a divisional of U.S. Ser. No. 07/978,582, filed Nov.19, 1992, now U.S. Pat. No. 5,352,667, issued Oct. 4, 1994.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel non-peptidic compounds havingterminal guanidino and carboxyl functional groups, to their preparationand to pharmaceutical compositions comprising them for treatment ofseveral pathological disorders.

The ability of various cell types to adhere to and to interact withother cells or with components of the extracellular matrix (ECM) isessential for maintaining cell functions and tissue integrity viasignalling between and within the communicating cells (Springer, 1990;Hynes, 1992; Shimizu et al., 1991). Cellular interactions with solubleor insoluble components of the plasma, the interstitial matrix or theECM, are carried out primarily via a family of cell-surface receptorsdesignated integrins that are present on most cell types, includinglymphocytes, tumor cells and platelets (Ruoslahti, 1991; Hynes, 1992).

The integrins are heterodimeric molecules consisting of an alpha (α) anda beta (β) subunits which are non-covalently linked. Eleven α and six βsubunits have been identified. The pairing of α and β subunits exhibitshigh fidelity in certain tissues and cell types but degenerates in othercases.

The integrins play an important role in integrating the ECM outside thecell with the actin-containing cytoskeleton inside the cell. They aretwo-headed: the extracellular portion is responsible for the binding ofadhesive proteins, in many cases recognizing the ARG-Gly-Asp (RGD; SEQID No:1) sequences within these ligands, and the intracellular portioninteracts with elements of the cytoskeleton.

The target epitope of several integrin receptors is the RGD sequence, acell adhesion motif shared by several matrix-associated adhesiveglycoproteins, such as fibronectin (FN), vitronectin (VN), fibrinogen,thrombospondin, and von Willebrand factor (Yamada & Kennedy, 1984;Hynes, 1992; Ruoslahti, 1988; D'Souza et al., 1991a).

The best characterized of these proteins is fibronectin, a large andabundant glycoprotein of extracellular matrices and plasma, which servesas a prototype cell adhesion molecule. Fibronectin is a multifunctionalprotein that supports cell attachment and spreading in eukaryotes andalso mediates bacterial cell adhesion. It binds to numerous cell surfaceand matrix constituents including glycosaminoglycans, heparin,proteoglycans, fibrin and collagen, and triggers a variety of cellularresponses (Hynes, 1990).

The tripeptide Arg-Gly-Asp (RGD) was identified as the minimal sequencewithin the central cell binding domain of fibronectin that mediates cellattachment. The RGD sequence is recognized by several receptors,including the αIIbβ3 (also designated GPIIb-IIIa), α3β1, α5β1 (alsodesignated VLA-3 and VLA-5 integrins, respectively) and most of theαv-containing integrins (Hynes, 1992; Elices et al., 1991; Shimizu etal., 1990). Following cell activation, these receptors mediateRGD-dependent cell-matrix adhesion or cell aggregation (Philips et al.,1991; D'Souza et al., 1991a; Adler et al., 1991). When present insolutions, peptides containing the RGD sequence compete with fibronectinand other RGD-containing matrix proteins for binding to their respectiveintegrin receptors and prevent cell adhesion (Springer, 1990; Ruoslahtiand Giancotti, 1989). When immobilized on a surface, short synthetic RGDpeptides mimic fibronectin cell-binding properties, but their affinityto their corresponding integrin is about 10² -10³ lower than that of thenative ligands (Humphries, et al., 1986).

The RGD motif is not restricted to fibronectin and in fact it is presentwithin more than hundred proteins. In some proteins, cell adhesiveactivity has been ascribed to the RGD sequence, whereas in most othersthe RGD sequence appears to be functionally silent. It was found to be acommon motif in cell adhesion molecules and it plays a crucial role inplatelet aggregation, the immune response, in cancer metastasis, cellmigration to tissues, infection of microbial pathogens, gastrulation inXenopus and Drosophila embryos. Several proteins, which were found tohave the sequence RGD expressed on their surface, promote cellattachment in vitro for no apparent physiological reason, alsoindicating the generality of this binding.

The functional activity of RGD peptides was demonstrated with a varietyof cell types. It is particularly significant that RGD peptides arecapable of inhibiting the binding of fibrinogen and other relatedproteins to platelets (small enucleated blood cells), and inhibitplatelet aggregation, the cell-cell interaction critical for thrombusformation. This observation indicated that RGD peptides could functionas antithrombotic agents.

European Patent Application published under No. EP 410539 describesfibrinogen receptor antagonists which are small cyclic hexapeptidescontaining the RGD sequence and are claimed to be useful in inhibitingplatelet aggregation. European Patent Application published under No. EP406428 describes synthetic cyclic peptides containing the RGD sequencewhich are cell adhesion inhibitors useful as platelet aggregationinhibitors and tumor metastasis suppressors. European Patent Applicationpublished under No. EP 394326 describes synthetic peptides whichincorporate the sequence RGD in a conformationally stabilised form andwhich may be utilized either for inhibiting binding of adhesionproteins, e.g. vitronectin, or for promoting cell adhesion, e.g. in vivouses such as coating of medical devices, including prostheses andimplants, or in vitro uses in coating of substrates such as cell culturesubstrates. European Patent Application published under No. EP 384362describes modified peptides useful as inhibitors of protein-plateletadhesion, cell-cell adhesion and platelet aggregation. InternationalApplication published under No. WO 9011297 describes adhesion peptidescomprising a biologically active site which is a cell attachmentpromoting binding site containing the RGD sequence, and a hydrophobicattachment domain, useful for facilitating the attachment of the peptideto solid substrates, e.g., in coating of prosthetic devices to beimplanted.

The physiological roles of RGD-mediated recognition may extend beyondthese biological processes. Pathogenic microorganisms may adhere toRGD-containing ECM glycoproteins. Thus, Trypanosoma cruzi adheres tofibronectin and peptides modeled from the fibronectin RGD cellattachment domain were shown to inhibit T. cruzi infection (Ouaissi etal., 1986).

Interestingly, several non-ECM related proteins contain the RGD orRGD-like molecules. Among these, the RGD sequence is also found in thetransactivation (tat) factor of human immunodeficiency virus type-I(HIV-1). The protein which regulates the viral replication also inducesother manifestations of the disease, e.g., Kaposi sarcoma. Soluble tatwas shown to bind to several integrins in an RGD-dependent manner (Vogelet al. 1992).

Peptides containing Arg-Ala-Asp (RAD; SEQ ID No:2), Arg-Glu-Asp (RED;SEQ ID No:3), Arg-Phe-Asp (RFD; SEQ ID No:4) and Arg-Tyr-Asp (RYD; SEQID No:5) sequences were postulated to interfere with immune functionsunrelated to integrins. The Arg-Ala-Asp-Ser (RADS; SEQ ID No:9),Arg-Phe-Asp-Ser (RFDS; SEQ ID No:10) and Arg-Tyr-Asp-Ser (RYDS; SEQ IDNo:11) sequences have been postulated to constitute functionaladhesiotopes of the CD4 or MHC-I and II molecules, respectively(Mazerolles et al., 1990). Human HLA-DR antigen, present on antigenpresenting cells, contains the sequence RYDS and is recognized by theT-cell CD4 antigen. Interference with the CD4-HLA-DR interaction mightresult in incomplete T cell activation.

Synthetic peptides derived from the human major histocompatibilitycomplex class II antigens (MHC-II) containing the peptide RFDS, and apeptide derived from the immunoglobulin-like amino-terminal domain ofthe T cell CD4 molecules containing the RADS peptide, were shown toexhibit specific inhibitory effect on antigen-induced HLAclass-II-restricted T cell proliferative responses and antibodysynthesis (Mazerolles et al, 1988).

The RYDS sequence-has been shown to mimic the RGD cell binding domain offibrinogen. RYD sequence is comprised as essential part of a CDR-3(complementarity-determining region) of a monoclonal antibody specificfor the binding site of the platelet integrin GPIIb-IIIa, specific forFN, fibrinogen, vitronectin etc. A 12-mer peptide derived from thisCDR-3, containing the RYDS site, inhibited RGD-dependent fibrinogenbinding to its GPIIb-IIIa receptor (Taub et al., 1989). StreptavidinRYD-sequence has also been shown to mimic the RGD sequence and mediateRGD-dependent cell binding and adhesion of the protein (Alon et al.,1990). Recently, REDV sequence of the alternatively spliced cell-bindingdomain of FN has been shown to be involved in FN-binding to its non-RGDdependent integrin receptor, α4β1 (Mould et al., 1991).

Moreover, the inverted peptide Ser-Asp-Gly-Arg (SDGR; SEQ ID No.12)containing the Asp-Gly-Arg (DGR; SEQ ID No:6) sequence was shown toinhibit spreading of BHK cells and chick embryo fibroblasts onvitronectin-coated substrates and on fibronectin-coated substrates.DGR-containing sequences have been suggested to comprise part of theligand-binding pocket in integrins, implicated in RGD recognition. Atany rate, they may interact with RGD-sequences on adhesive proteins andblock or inhibit their interactions with integrins (Yamada and Kennedy,1987).

The use of peptidic RGD analogues presents several drawbacks, mainly thecleavage of the peptidic bond by proteolytic enzymes in vivo. It wouldtherefore be of great advantage to derive functional mimetics resistantto proteolytic digestion to be used as useful tools for interfering withbiologic interactions dependent on RGD recognition, such asintegrin-mediated cell functions.

SUMMARY OF THE INVENTION

It has now been found according to the present invention that certainnon-peptidic compounds comprising a guanidino and a carboxyl terminalgroups with a spacer sequence of 11 atoms between them, are effectiveinhibitors of cellular or molecular interactions which depend on RXD orDGR recognition, wherein X is G (gly), E (glu), Y (tyr), A (ala) or F(phe). These RXD and DGR analogues are herein referred to as "RXDsurrogates".

The present invention thus relates to non-peptidic compounds having nosequence of natural α-amino acids and comprising a guanidino and acarboxyl terminal functional groups spaced by a sequence of 11 atoms, atleast 5 of which are carbon atoms, and to salts thereof, which arecapable of inhibiting cell adhesion.

In one embodiment, the compounds of the invention correspond to thegeneral formula ##STR2## wherein A is a chain of 9 atoms, at least 3 ofwhich are carbon atoms, the remainder being heteroatoms, such asnitrogen, oxygen and/or sulfur atoms. The 9-atom chain A may besaturated or unsaturated, substituted or unsubstituted, and may includecarbocyclic or heterocyclic radicals comprising 1 or more atoms of the Achain as members of the ring.

The invention further relates to methods for the preparation of thenon-peptidic compounds of the invention.

The RXD surrogates of the invention have various applications related totheir inhibition of biological interactions dependent on RXD and DGRrecognition, particularly integrin-mediated RGD-dependent interactions.Thus the invention also relates to pharmaceutical compositionscomprising the RXD surrogates for the treatment of several disorders,such as thrombosis, metastasis, autoimmune diseases and other immuneresponses such as allergy, graft versus host and host versus graftreactions, and inhibition of scar-tissue formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of compounds according to theinvention identified as SF-6,5, AC-15, AC-4 and AC-14, and compoundsused for comparison identified as SF-6,6 and SFN-70.

FIG. 2 depicts the chemical structure of compounds according to theinvention identified as NS-8, NS-11 and NS-15.

FIG. 3 shows a dose-dependent curve of inhibition of plateletaggregation by compounds SF-6,5 (filled circles) and SF-6,6 (emptycircles).

FIG. 4 illustrates inhibition of platelet aggregation by the compoundsof the invention SF-6,5 and AC-15, and the compounds RGD,Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP; SEQ ID No:13) and SF-6,6 forcomparison.

FIG. 5 illustrates platelet aggregation inhibition by the compounds ofthe invention NS-8 (empty triangles)i, NS-11 (filled squares) and NS-15(filled circles) in comparison to peptide ARG-Gly-Asp-Ser (RGDS; SEQ IDNo:8) (crossed squares).

FIG. 6 illustrates inhibition of the binding capacity of anti-GPIIb-IIIamonoclonal antibody (PAC-1) to platelets by the compound of theinvention SF-6,5 (empty triangles), in comparison to compound SF-6,6(filled circles) and peptides GRGDSPK (empty circles) andGly-Arg-Gly-Glu-Ser-Pro (GRGESP; SEQ ID No:14) (filled triangles).

DETAILED DESCRIPTION OF THE INVENTION

In designing the non-peptidic ARG-Gly-Asp (RGD; SEQ ID No:1) surrogatesaccording to the present invention, it was taken into consideration thatthe major contribution to the binding affinity of the knownRGD-containing peptides to their putative sites on integrins depends onthe guanidinium and carboxylate groups of the Arg and Asp moieties,respectively, based on structure-function studies demonstrating theindispensable role of the Arg and Asp residues for integrin recognition(D'Souza et al., 1991b), and the fact that an adequate atomic spacingbetween these two functional groups seems to be obligatory, based on theevidence that ARG-Gly-Glu (RGE; SEQ ID No:7)-containing peptides lackintegrin specificity and do not bind integrins or bind integrins withmuch lower affinity than the RGD ligands (D'Souza et al. 1991b; Shimizuet al., 1990). Furthermore, it was considered that even a relativelyflexible chain molecule with appropriate functionalities at the requiredatomic distances can exhibit functional effects resembling those ofRGD-containing peptides.

As used herein the term "RXD surrogates" refers to novel non-peptidiccompounds having no sequence of natural α-amino acid residues andcomprising a guanidino and a carboxyl terminal groups spaced by a chainof 11 atoms, at least 5 of which are carbon atoms, and the remainder arecarbon or heteroatoms, such as nitrogen, oxygen and/or sulfur atoms, aswell as to salts thereof.

The two carbon atoms adjacent to the terminal functional guanidino andcarboxy groups are preferably not substituted as shown in Formula Iherein. The remaining 9-atom chain may be saturated or unsaturated,substituted or unsubstituted.

The substituents in the spacer chain include, but are not limited to,radicals such as halogen, amino, oxo, thioxo, imino, hydrocarbyl,heterocyclic, carboxyl and thiocarboxyl and esters thereof, carboxamido,thiocarboxamido, carbamoyl, thiocarbamoyl, hydroxy, and ethers andesters thereof, mercapto and ethers and esters thereof. All thesubstituents having a hydrogen atom may be further substituted, e.g. bya hydrocarbyl or heterocyclic radical. The esters and ethers hereincomprise aliphatic, aromatic and heterocyclic residues, preferablyhydrocarbyl and heterocyclic residues that may be further substituted asindicated above for the spacer chain. Esters of hydroxyl groups may beformed also with inorganic acids, e.g., phosphoric acid. In addition,one or more atoms of the spacer chain may form part of a carbocyclic orheterocyclic ring having at least 3 members.

The term "hydrocarbyl" herein refers to C₁ -C₁₅ saturated andunsaturated radicals selected from aliphatic, cycloaliphatic and arylradicals, such as alkyl, alkenyl, cycloalkyl and aryl radicals.Preferred hydrocarbyl radicals are C₁ -C₈, more preferably C₁ -C₄ alkylradicals, and phenyl.

The term "heterocyclic" herein refers to saturated and unsaturated 3-8,preferably 5-7 membered heterocyclic radicals containing one or more N,O and/or S atoms, such as piperidyl and pyridyl.

For use in therapeutics, the compound should be soluble in water and anysubstituent resulting in a soluble compound is encompassed by theinvention. Examples of such substituents are oxo groups, thus forming--CO--NH- or --NH--CO-groups within the chain, and carboxy and/or aminogroups.

A preferred series of compounds according to the invention includescompounds having one or more --CO--NH-residues and may be represented bythe following formulas: ##STR3## wherein in formulas Ia and Ib n is atleast 1 and at most 8, and in formulas Ic and Id each of x, n and m isat least 1 and the sum of x+m+n is 7. Illustrative compounds of thisseries are the compounds herein designated SF-6,5 and AC-15, whoseformulas are depicted in FIG. 1, and are compounds of formula Ia whereinn is 5 and 4, respectively, and the compounds herein designated AC-4 andAC-14, whose formulas are depicted in FIG. 1 and are compounds offormula Ic wherein x is 4, n is 1 and m is 2 or x is 3 and each of n andm is 2, respectively.

Other compounds according to the invention are illustrated by thefollowing formulas: ##STR4##

Another series of preferred compounds according to the inventioncomprises compounds having one or more --CO--NH-residues and acarbocyclic, particularly a phenyl ring, or heterocyclic, particularly apiperidine ring, as part of the A chain. Whenever one or more atoms ofthe A chain form part of such a ring, they are comprised according tothe invention within the shortest chain of the ring between atoms of theopen chain. These compounds may be represented by the followingformulas: ##STR5## wherein each of n and m is at least 1 and the sum ofn+m is 6 in formula IIIc, 5 in formula IIId, 4 in formulas IIIa and IIIeand 3 in formula IIIb. Illustrative compounds of this series arecompounds NS-8, NS-11 and NS-15 depicted in FIG. 2, which are compoundsof formula IIIc (n=4, m=2), IIId (n=3, m=2) and IIIa (n=m=2),respectively.

Another series of preferred compounds are those of the followingformulas: ##STR6## wherein n is at least 1 and at most 7 in formulas IVcand IVd, 8 in formulas IVb and IVe, and 9 in formula IVa.

The invention further comprises salts of the surrogates of the inventionderived from organic or inorganic bases.

The compounds of formula Ia are prepared by a process comprising of thefollowing steps:

(a) coupling an N-- protected aminocarboxylic acid of the formulaZNH--(CH₂)_(n) --COOH (wherein Z is a protecting group, such asN-t-butyloxycarbonyl, herein N-t-Boc) with an alkyl ester of anaminocarboxylic acid of the formula H₂ N--(CH₂)_(9-n) --COOR, wherein Ris lower alkyl, using standard procedure, for example, with1,3-dicyclohexylcarbodiimide and 1-hydroxy-benzotriazole orN-hydroxysuccinimide;

(b) removing the protecting group from the obtained compound of theformula ZNH--(CH₂)_(n) --CO--NH--(CH₂)_(9-n) --COOR, for example, withtrifluoroacetic acid, resulting in a compound of the formula H₂N--(CH₂)_(n) --CO--NH--(CH₂)_(9-n) --COOR; and

(c) converting the free amino group to a guanidino group, for example,by reaction with 3,5-dimethylpyrazole 1-carboxamidine nitrate, withconcomitant removal of the ester group R.

According to the above process, the compounds SF-6,5 and AC-15 wereprepared by coupling methyl 6-aminohexanoate withN-t-butyloxycarbonyl-5-aminopentanoic acid, or methyl 5-aminopentanoatewith N-t-butyloxycarbonyl-6-aminohexanoic acid, respectively, using1,3-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole indichloromethane. The butyloxycarbonyl protecting group was then removedby 50% trifluoroacetic acid in dichloromethane, and the amine wasconverted to guanidine using 3,5-dimethylpyrazole 1-carboxamidinenitrate at pH 9.5. The methyl group was removed under the reactionconditions.

Compounds of formula Ic are prepared by stepwise synthesis on aMerrifield resin according to standard procedure (Barany and Merrifield,1980), Thus an N-t-Boc-omega-amino acid is prepared and coupled to achloromethylated polystyrene 1% divinyl benzene by the cesium saltmethod. Coupling on the polymer may be carried out manually with 2 foldexcess of the N-t-Boc-omega-amino acid with an equimolar mixture ofN,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole as reagents.Deprotection by 50% trifluoroacetic acid in methylene chloride andcoupling to the next N-t-Boc-omega-amino acid under the same conditionsyields the final product coupled to the polymer. Deprotection andcleavage from the resin is achieved by treatment with anhydrous HF. Thecrude product is extracted in 50% acetic acid and lyophilized.Conversion of the amino to the guanidino group is carried out asdescribed above for the preparation of compound Ia. The final product ispurified by reverse phase chromatography followed by preparative HPLCpurification.

Compounds of formula Ib are prepared by a stepwise synthesis comprisingcoupling of a monoprotected diamine of the formula ZNH--(CH₂)_(n) --NH₂with a monoalkyl ester of a dicarboxylic acid of the formulaHOOC--(CH₂)_(9-n) --COOR, wherein R is lower alkyl, and removal of theprotecting group and conversion of the amino to the guanidino group, asdescribed above in steps (b) and (c) for the preparation of compounds offormula Ia. For example, when n is 3,N-monobenzyloxycarbonyl-propanediamine is coupled with the monomethylester of suberic acid.

Compounds of formula Id are prepared by first coupling anaminocarboxylic acid of the formula HOOC--(CH₂)_(n) --NH₂ with amonoester of a dicarboxylic acid of the formula HOOC--(CH₂)_(m) --COOR,wherein R is lower alkyl, followed by further coupling with amonoprotected diamine of the formula ZNH--(CH₂)_(x) --NH₂, removal ofthe protecting group and conversion of the amino to guanidino group asdescribed above. When x is 3 and n=m=2, β-alanine is coupled withmonomethylsuccinate and the resulting compound is coupled withN-monobenzyloxycarbonyl-propanediamine.

Compounds substituted by amino or carboxyl groups, such as those offormulas IIa, IIb and IIc above, are prepared by stepwise synthesis on aMerrifield resin, using the appropriate substituted aminocarboxylic acidand/or monoprotected diamine (comp. IIc).

Compounds of the formulas IIIa-e are prepared by stepwise synthesis on aMerrifield resin according to standard procedure as described above forpreparation of compounds of formula Ic.

Compounds of formula IVa are prepared by a process comprising couplingof a monoprotected diamine of the formula ZNH--(CH₂)_(n) --NH₂ (whereinZ is a protecting group, such as N-t-Boc or benzyloxycarbonyl) with analkyl ester of an omega-bromocarboxylic acid of the formula Br--(CH₂)₁₀--_(n) --COOR wherein R is lower alkyl, using standard procedure, forexample, dimethyl formamide as solvent and triethyl amine as base.Removal of the protecting group and conversion of the primary amine tothe guanidino group is carried out as decribed above for the preparationof compounds of the formula Ia. Thus, to prepare a compound wherein n is5, N-monobenzyloxycarbonyl-pentanediamine is coupled with the methylester of 5-bromovaleric acid.

Compounds of formula IVb are prepared by a process comprising couplingof an N-protected amino alcohol of the formula ZNH--(CH₂)_(n) --OH witha monoester of a dicarboxylic acid of the formula HOOC--(CH₂)₉ --_(n)--COOR using 1,3-dicyclohexylcarbodiimide as the coupling agent. Removalof the protecting group and conversion of the primary amine to theguanidino group is carried out as described above for the preparation ofcompounds of the formula Ia. Thus to prepare a compound wherein n is 5,N-t-butyloxycarbonylamino pentanol is coupled with monomethyl adipate.

Compounds of formula IVc are prepared by a process comprising reactionof an omega-bromocarboxylic acid ester of the formula Br--(CH₂)_(8-n)--COOR and sodium cyanate, thus forming an omega-isocyanatocarboxylicacid alkyl ester of the formula OCN--(CH₂)_(8-n) --COOR acid. Furtherreaction with a monoprotected diamine of the formula ZNH--(CH₂)_(n)--NH₂ produces the protected urea of the formula ZNH--(CH₂)_(n)--HNCONH(CH₂)_(8-n) --COOR. Removal of the protecting group andconversion of the primary amine to the guanidino group is carried out asdescribed above for the preparation of compounds of the formula Ia.Thus, to prepare a compound wherein n is 3, 5-bromovaleric acid methylester is converted to 5-isocyanatovaleric acid methyl ester which isthen reacted with N-monobenzyloxycarbonyl-propanediamine.

Compounds of formula IVd are prepared by reacting anomega-isocyanatocarboxylic acid alkyl ester of the formulaOCN--(CH₂)_(8-n) --COOR, prepared as above, with an N-protected aminoalcohol of the formula ZNH--(CH₂)_(n) --OH to form the protectedcarbamate of the formula ZNH--(CH₂)_(n) --OCONH(CH₂)_(8-n) --COOR.Removal of the protecting group and conversion of the primary amine tothe guanidino group is carried out as described above for thepreparation of compounds of the formula Ia. For example, to prepare acompound wherein n is 3, 5-isocyanatovaleric acid methyl ester isreacted with 3-N-t-butyloxycarbonylamino propanol.

Compounds of formula IVe are prepared by a process comprising thefollowing steps: (a) an omega-acetylthiocarboxylic acid alkyl ester ofthe formula CH₃ COS--(CH₂)_(9-n) --COOR is prepared by reaction of anomegabromocarboxylic acid alkyl ester of the formula Br--(CH₂)_(9-n)--COOR with sodium thioacetate, (b) an N-protected amino alcohol of theformula ZNH--(CH₂)_(n) --OH is converted first to its tosylate byreaction with p-toluene sulfonyl chloride in pyridine and then byreaction with sodium thioacetate, to the thioacetate of the formulaZNH--(CH₂)_(n) --SCOCH₃, (c) the acetate groups of the compoundsprepared in steps (a) and (b) are removed under basic conditions, e.g.,sodium carbonate, producing the corresponding thiol compounds; and (d)the asymmetrical disulfide of the formula ZNH--(CH₂)_(n) SS(CH₂)₉ --_(n)--COOR is then formed by reaction of the two thiols of step (c) usingdiethyl azadicarboxylate as oxidation agent. For example, to prepare acompound wherein n is 4, 5-bromovaleric acid methyl ester is convertedto the protected thiol by reaction with sodium thioacetate, andN-t-butyloxycarbonyl-4-aminobutanol is converted to the correspondingtosylate followed by substitution, with sodium thioacetate. The acetategroups are then removed under basic conditions and the resultingN-t-butyloxycarbonyl-4-aminobutanethiol is added to a solutioncontaining the resulting 5-mercaptovaleric acid methyl ester and diethylazadiacarboxylate.

All the final compounds were purified on preparative RP-18 columns andwere judged pure by thin layer chromatography (single spot) and ¹ H NMRspectroscopy. Compounds were characterized by ¹ H NMR and FAB MSspectroscopy which were consistent with the assigned structures.

The RXD surrogates of the invention can inhibit biological interactionswhich are dependent on RXD recognition. Examples of versatilerecognition processes mediated by the RXD pattern encompassed by thepresent invention include, but are not limited to, cellular andmolecular interactions involving the ARG-Gly-Asp (RGD; SEQ ID No:1),Arg-Tyr-Asp (RYD; SEQ ID No:5), Arg-Glu-Asp (RED; SEQ ID No:3),Arg-Ala-Asp (RAD; SEQ ID No:2), Arg-Phe-Asp (RFD; SEQ ID No:4) andAsp-Gly-Arg (DGR; SEQ ID No:6) sequences, in particularintegrin-mediated RGD-dependent interactions.

The non-peptidic RXD surrogates of the invention inhibit cell adhesion.As used herein the term "cell adhesion" encompasses any of the followinginteractions: (a) cell-cell adhesion, illustrated by plateletaggregation; (b) cell adhesion to glycoproteins of the serum or of theECM, illustrated by adhesion of lymphocytes and metastatic tumor cellsto RGD-containing glycoproteins of ECM; (c) pathogenic organismsadherence to RGD-containing glycoproteins of ECM, illustrated byadhesion of Trypanosoma cruzi to fibronectin; and (d) cell adhesion toRGD-containing non-ECM proteins, illustrated by adhesion of lymphocytesto the tat factor of HIV-1.

The surrogates of the invention are capable of inhibiting plateletaggregation. The interaction of lymphocytes and tumor cells with FNpresent in the interstitial matrix and on cell surfaces have beenpostulated to play a major role in cell adhesion and migration. In viewof the ability of RGD surrogates to interfere with platelet aggregation,it was tested whether they could also competitively inhibit Tlymphocytes and tumor cells binding to FN or VN; such inhibition hasbeen shown to be strongly associated with the RGD sequence on both ofthose adhesive proteins (van Seventer et al, 1991; Shimizu et al, 1990;Adler et al, 1991; Ruoslahti et al, 1989). The answer was positive andthe results are shown in Tables 1A and 1B hereinbelow.

As inhibitors of cell adhesion the compounds of the invention caninhibit adhesion of cancer cells to fibronectin and vitronectin, andthus can prevent metastasis. They also block lymphocyte migration totissues and thus inhibit several immune disorders, such as allergy,which depends on immune cells. They also inhibit platelet aggregationand thus can be used in the prevention and/or treatment of plateletthrombosis, thromboembolism, reocclusion after angioplasty of coronaryand other arteries and myocardial infarction. In addition, thesurrogates inhibit key lymphocyte interaction with certainantigen-presenting cells and thus inhibit T cell activation, beinguseful in the treatment of autoimmune diseases. Through competition withfibronectin recognition by fibroblasts implicated in the fibrosisprocess, the surrogates inhibit scar tissue formation at a very earlystage, being useful in wound healing process.

Thus in one preferred embodiment, the compounds of the invention are RGDsurrogates and will inhibit both cellular and molecular interactionswhich are RGD dependent. In this respect, the compounds are useful inthe treatment of a series of disorders, including thrombosis, autoimmunediseases, metastasis, immune disorders such as allergy, graft versushost and host versus graft reactions, and in wound healing in theinhibition of scar formation.

The compounds of the invention can be administered to patients by anysuitable route including oral and parenteral routes, e.g., intravenous,subcutaneous or intramuscular injection. An effective but essentiallynon-toxic quantity of the compound will be employed in the treatment.Effective amounts may be within the range of 0.01 to 1 mg/kg, preferably0.5 mg/kg on a regimen in single or several daily doses.

The invention further provides a pharmaceutical composition comprisingas active ingredient a surrogate according to the invention and apharmaceutically acceptable carrier. The compositions may be in the formof tablet, capsule, solution or suspension containing from about 0.7 to70 mg per unit of dosage of an active compound of the invention ormixtures thereof. The compounds may be compounded in conventional mannerwith a physiologically acceptable vehicle or carrier, excipient binder,preservative, stabilizer, etc. For example, injections for intravenousadministration may be prepared in saline, at a pH level of e.g. 7.4,suitable for achieving inhibition of platelet aggregation.

In another embodiment the RGD surrogates of the invention can be used aspromoters of cell adhesion to a surface, for example, in vivo uses suchas coating of medical devices, including prostheses or implants, e.g.,vascular implants thus facilitating the attachment of cells thereto.They may also be used in vitro for coating of substrates, e.g. cellculture substrates to promote cell adhesion.

The following examples are intended to illustrate, by way of example,the principles of the invention, without limiting it thereto.

EXAMPLES Example 1. Preparation of 6-aza-7-oxo-12-guanidino-dodecanoicacid [SF-6,5]

1.1 Preparation of N-t-Boc-6-aminohexanoic acid [compound 2]

Di-t-butylpyrocarbonate (8.31 g, 38 mmol) was added to a solution of6-aminohexanoic acid [compound 1] (5 g, 38 mmol) and sodium hydroxide(38 ml, 2N solution) in dioxane (30 ml).

The solution was stirred at room temperature for 4 hours, then it wasacidified with 2N HCl and poured into ether (100 ml). The phases wereseparated, the ether layer was dried and removed under reduced pressure,whereupon compound [2] was obtained and used in the next step withoutfurther purification.

1.2 Preparation of methyl 5-aminopentanoate [compound 4]

5-Aminopentanoic acid [compound 3] (5 g, 41 mmol) was added to asaturated HCl solution in dry methanol (100 ml). The solution was leftfor 3 h at room temperature and the methanol was removed under reducedpressure. The residue was redissolved in a minimum amount of methanoland precipitated by addition of ether, whereupon the white crystalinecompound [4] was obtained, filtered, washed with ether, dried in vacuoand used in the next step without further purification.

1.3 Coupling of compounds [2] and [4] by the active ester method.

An active ester of compound [2] was generated by addition of1,3-dicyclohexylcarbodiimide (DCC) (490 mg, 2.37 mmol) to a solutioncontaining compound [2] (500 mg, 2.16 mmol) and N-hydroxysuccinimide(273 mg, 2.37 mmol) in CH₂ Cl₂ /THF 1:1 (v/v) solution (7 ml) and leftovernight at room temperature. The resulting active ester solution wasfiltered to remove dicyclohexylurea (DCU) generated in the reaction andwashed with dry CH₂ Cl₂. A solution of compound [4] containing triethylamine (to neutralize the amine hydrochloride) in DMF (5 ml) was thenadded to the active ester of compound [2] solution. After 5 h, thesolution was poured into 5% aqueous sodium bicarbonate (100 ml) and theproduct was extracted with ether. The ether was dried over Na₂ SO₄ andremoved under reduced pressure, whereupon the crude product [5] wasobtained pure enough for the next step.

1.4 Removal of the N-t-Boc protecting group

The crude product [5] was dissolved in CH₂ Cl₂ :TFA (trifluoroaceticacid) 1:1 v/v at 0° C. After 30 min, the solution was allowed to warm toroom temperature for 30 min. The solvents were removed under reducedpressure, the crude product was dissolved in water and washed withether. The water was removed under reduced pressure, whereupon the crudeproduct with a free amino group was obtained and used in the next stepwithout further purification.

1.5 Preparation of the compound SF-6,5

3,5,Dimethylpyrazole 1-carboxamidine nitrate (175 mg, 0.87 mmol) wasadded to a solution containing the deprotected amine (200 g, 0.87 mmol)obtained in step 1.4 in ethanol (5 ml), and NaOH (2N) was added to thesolution to bring the pH to 9.5. The solution was stirred overnight at50° C., the solvent was removed under reduced pressure and the crudeproduct SF-6,5 was purified by reverse phase chromatography, followed byHPLC using RP-18 column. Purity was checked by NMR.

NMR: 3.18(t, J=6.54 Hz, 2 H); 3.16(t, J=6.90 Hz, 2 H); 2.39(t, J=7.19, 2H); 2.23(t, J=7.21 Hz, 2 H); 1.64-1.49(M, 8 H); 1.33 (tt, 2 H). FAB-MS(m/e): 273.3 M+1).

Example 2. Preparation of 7-aza-8-oxo-12-guanidino-dodecanoic acid[AC-15]

The compound AC-15 was prepared similarly to compound SF-6,5 asdescribed in Example 1, but using as starting materials methyl5-aminopentanoate and N-t-butyloxycarbonyl 6-aminohexanoic acid. Puritywas checked by NMR.

NMR: 2.98(t, J=6.45 Hz, 2 H); 2.97(t, J=6.69 Hz, 2 H); 2.06(t, J=6.79Hz, 2 H); 1.97(t, J=7.38 Hz, 2 H); 1.49-1,24(m, 8 H); 1.16-1.05(tt,2 H).FAB-MS: (m/e): 273.3 (M+1).

Example 3. Preparation of compound SF-6,6

This compound has an extra methylene group in the spacer chain and wasused for comparison with the compounds of the invention. It was preparedsimilarly to compound SF-6,5 as described in Example 1, but using asstarting compounds N-t-Boc-6-aminohexanoic acid and methyl6-aminohexanoate.

NMR: 3.03(t, J=6.75 Hz 2 H); 3.02(t, J=6.96 Hz, 2 H); 2.23(t, J=7.38, 2H); 2.09(t, J=7.28 Hz, 2 H); 1.46(M, 6 H); 1.37(Q, 2 H); 119(tt,4 H).FAB-MS (m/e): 287.3(M+1).

Example 4. Preparation of compound SFN-70

This compound has a similar structure to compound SF-6,5, but instead ofa terminal guanidino group it has a primary amino group. It was preparedby hydrolysis of the appropriate ester from Example 1.4 in aqueous base,and used for comparison with the compounds of the invention.

Example 5. Preparation of 4,8-diaza-5,9-dioxo-12-guanidino-dodecanoicacid [AC-14]

N-t-Boc-β-alanine was prepared as described in Example 1 for the6-aminohexanoic acid and was coupled to a chloromethylated polystyrene1% divinyl benzene by the cesium salt method. Thus t-Boc-β-alanine (1.73g, 0.01 mol) was dissolved in water (10 ml) and the pH was adjusted to7.0 by adding a solution of 1M Cs₂ CO₃. The solvent was removed underreduced pressure and the residue was dried in vacuo over P₂ O₅. The drysalt was dissolved in DMF and was added to the polymer. The mixture waskept at 50° for 12 h with occasional shaking. The solvent was filteredoff and the polymer was washed successively with DMF, DMF:water 9:1mixture and ethanol and was dried in vacuum. Coupling on the polymer wascarried out manually. Thus after deprotection with 50% trifluoroaceticacid in methylene chloride, coupling to N-t-Boc-β-alanine was performedwith 2 fold excess of the protected amino acids with an equimolarmixture of 1,3-dicyclohexyl-carbodimide and 1-hydroxybenzotriazole asreagents. Deprotection and coupling to N-t-Boc-gamma-aminobutyric acidunder the same conditions gave the final product coupled to the polymer.Deprotection and cleavage from resin was achieved by treatment withanhydrous HF.

The crude product was extracted in 50% acetic acid and lyophilized.Conversion of the amino to the guanidino group was carried out asdescribed for the preparation of compound SF-6,5 in Example 1. The finalproduct was purified by reverse phase chromatography followed bypreparative HPLC purification.

AC-14, NMR;3.58(t, J=6.41 Hz, 2 h); 3.52(t, J=6.74 Hz, 2 H); 2.56(t,J=6.42 Hz, 2 H); 2.53(t, J=6.72 Hz, 2 H); 2.44(t, J=7.25 Hz, 2 H); 2.00(Q, 2 H). FAB-MS: (m/e): 288.3 (M+1).

Example 6. Preparation of 4,7-diaza-5,8-dioxo-12-guanidino-dodecanoicacid [compound AC-4]

This compound corresponds to the formula (Ic) where x is 4, n is 1 and mis 2. It was prepared similarly to compound AC-14 as described inExample 5.

NMR; 3.70(S, 2 H); 3.31(t, J=6.3 Hz, 2 H); 3.04(t, J=6.6 Hz, 2 H);2.44(t, J=6.4 Hz, 2 H); 2.19(t, J=6.7 Hz, 2 H); 1.46(M, 4 H). FAB-MS:(m/e): 288.3 (M+1).

Example 7. Preparation of4,8-diaza-5,9-dioxo-7-carboxy-12-guanidino-dodecanoic acid [compoundIIa]

Compound [IIa] was prepared on a Merrifield resin starting withN-t-Boc-β-alanine coupled to the polymer as in Example 5. Couplingon-the polymer was carried out manually as in Example 5, first couplingto α-benzyl N-t-Boc-aspartic acid followed by coupling toN-t-Boc-gamma-aminobutyric acid. Deprotection, cleavage from the resinand conversion of the amino to the guanidino group were carried out asin Example 5.

Example 8. Preparation of4,8-diaza-5,9-dioxo-3-carboxy-12-guanidino-dodecanoic acid [compoundIIb]

For preparation of the compound [IIb], α-benzyl N-t-Boc-aspartic acidwas coupled to the Merrifield resin by the same method described inExample 5. Stepwise synthesis by addition of N-t-Boc-β-alanine followedby coupling to N-t-Boc-gamma-aminobutyric acid produced afterdeprotection, cleavage, conversion of the amino to the guanidino groupand reverse phase chromatography, the compound [IIb].

Example 9. Preparation of4,9-diaza-5,8-dioxo-7-amino-12-guanidino-dodecanoic acid [compound IIc]

Compound IIc was prepared by stepwise synthesis in solution startingwith benzyl β-alanine using the active ester method as in the Merrifieldmethod. Thus benzyl β-alanine was coupled to α-benzyl N-t-Boc-asparticacid and the product was deprotected in TFA:CH₂ Cl₂ 1:1 as in Example1.4. It was then coupled to monobenzyloxycarbonyl 1,3-propanediamine.Deprotection, followed by conversion of the amino to the guanidino groupas described in Example 1.5, gave compound IIc which was purified byreverse phase chromatography.

Example 10. Preparation of 9-aza-8-oxo-12-guanidino-dodecanoic acid[compound Ib, wherein n=3]

10.1 The diprotected diamine H₂ N--(CH₂)₃ --NH_(z) was prepared byadding benzyloxycarbonylchloride (1.2 mole) with 2 equivalents of 1NNaOH to a solution of the diamine (1 mole) in water (500 ml). Theproduct was washed with water and hexane, dried over P₂ O₅, andrecrystallized from ethanol.

10.2 The monoprotected diamine was prepared by heating under reflux asolution of (0.06 mole) of the diprotected diamine in glacial aceticacid (100 ml) and concentrated HCl (10 ml, 0.12 mole) for 1 h andallowing to stand at room temperature overnight. The dihydrochloride ofthe diamine was crystallized and was filtered. Themonobenzyloxycarbonylpropanediamine was precipitated from the filtrateby the addition of ether. It was filtered, washed with ether and driedand was found to be pure enough for use in the next step.

10.3 1,3-Dicyclohexylcarbodiimide (490 mg), 2.37 mmol) was added to asolution containing monomethyl suberate (2.16 mmol) andN-hydroxysuccinimide (2.37 mmol) in CH₂ Cl₂ /THF 1:1 (v/v) solution (7ml). The reaction was left overnight at room temperature, the solutionwas filtered to remove DCU and washed with dry CH₂ Cl₂.Monobenzyloxycarbonylpropanediamine of step 10.2 in solution containingtriethyl amine (to neutralize the amine hydrochloride) in DMF (5 ml) wasthen added to the active ester solution. After 5 h, the solution waspoured into 5% aqueous sodium bicarbonate (100 ml) and the product wasextracted with ether. The ether was dried over Na₂ SO₄ and was removedunder reduced pressure. The crude coupling product was pure enough forthe next step.

10.4 The benzyloxycarbonyl protecting group was removed and the crudeamine was converted to the guanidine (the methyl ester group was cleavedunder the reation conditions), as described in previous examples.Purification by reverse phase chromatography afforded the title product.

Example 11 Preparation of 5,9-diaza-4,8-dioxo-12-guanidino-dodecanoicacid [compound Id, Where x=3 and n=m=2]

1,3-Dicyclohexylcarbodiimide (490 mg, 2.37 mmol) was added to a solutioncontaining monomethyl succinate (2.16 mmol) and N-hydroxysuccinimide(2.37 mmol) in CH₂ Cl₂ /THF 1:1 (v/v) solution (7 ml). The reaction wasleft overnight at room temperature. The solution was filtered to removeDCU and washed with dry CH₂ Cl₂. β-alanine solution in DMF (5 ml) wasthen added to the active ester solution. After 5 h, the solution waspoured into water (100 ml) and the product was extracted with ether. Theether was dried over Na₂ SO₄ and removed under reduced pressure. Thecrude coupling product was pure enough for the next step. Coupling withmonobenzyloxycarbonyl-propanediamine, deprotection, conversion of theamine to the guanidine and purification was done as in previousexamples.

Example 12. Preparation of compound NS-11

N-t-Boc-β-alanine was prepared as described for theN-t-Boc-6-aminohexanoic acid in Example 1 above and was coupled to achloromethylated polystyrene 1% divinyl-benzene by the cesium saltmethod. Thus t-Boc-β-alanine (1.73 g, 0.01 mol) was dissolved in water(10 ml) and the pH was adjusted to 7.0 by adding a solution of 1M Cs₂CO₃. The solvent was removed under reduced pressure and the residue wasdried in vacuo over P₂ O₅. The dry salt was dissolved in DMF and wasadded to the polymer. The mixture was kept at 50° C. for 12 h withoccasional shaking. The solvent was filtered off and the polymer waswashed successively with DMF, DMF:water 9:1 mixture and ethanol and wasdried in vacuo. Coupling on the polymer was carried out manually, firstwith N-t-Boc nipecotic acid (prepared as described for the6-aminohexanoic acid) followed by N-t-Boc α-aminobutyric acid. Allcouplings were performed with 3 fold excess of protected amino acidderivatives with an equimolar mixture of N,N'-dicyclohexylcarbodiimideand 1-hydroxybenzotriazole as reagents. Deprotection and cleavage fromresin was achieved by treatment with anhydrous HF. The crude product wasextracted in 50% acetic acid and lyophilized. Conversion of the amino tothe guanidino group was carried out as described above. The finalproduct was purified by reverse phase chromatography followed bypreparative HPLC purification.

Example 13. Preparation of compounds NS-8 and NS-15

13.1 Compound NS-8 was prepared on a Merrifield resin (Sigma) startingwith N-t-Boc-β-alanine coupled to the polymer as in Example 5. Couplingson the polymer were carried out manually as in Example 5, coupling firstto N-t-butyloxycarbonyl pipecolic acid followed by coupling ofN-t-butyloxycarbonyl 5-aminovaleric acid. Deprotection, cleavage fromthe resin and conversion of the amino to the guanidinium group werecarried out as in Example 5.

13.2 Compound NS-15 was prepared on a Merrifield resin (Sigma) startingwith N-t-Boc-β-alanine coupled to the polymer as in Example 5. Couplingson the polymer were carried out manually as in Example 5, coupling firstto N-t-butyloxycarbonyl 3-aminobenzoic acid followed by coupling ofN-t-butyloxycarbonyl β-alanine. Deprotection, cleavage from the resinand conversion of the amino to the guanidinium group were carried out asin Example 5.

All compounds were characterized by ¹ H-NMR and FAB-MS which wereconsistent with the assigned structures depicted in FIG. 2.

Example 14. Treatment with trypsin

Compound SF-6,5 and the Gly-Arg-Gly-Asp-Ser (GRGDS; SEQ ID No:15)peptide (50 μg in 100 μl PBS) were incubated at 37° C. and exposed to0.25% trypsin (Gibco; 50 μl in modified Puck's buffer). Aliquots weretaken after 5, 30 and 60 min and monitored by HPLC at 220 nm. CompoundSF-6,5 was found intact after 60 min while the GRGDS peptide wascompletely hydrolized after 5 min.

Example 15. Inhibition of T cell adhesion to ECM protein

To examine the adhesive properties of the T cells, 1 μg/50 μl/well ofeither fibronectin (FN) (Sigma), the 120 kD cell attachment fragment ofFN (Telios Pharm. Inc. San Diego, Calif.), or laminin (LN) (Sigma), wereadded to 96-flat bottom microtiter-wells for 12 h. Unbound proteins werethen washed away and remaining binding sites were blocked with 0.1%bovine serum albumin (BSA) added to the wells for 2 h and washed. CD4+ Tcells were purified from peripheral blood mononuclear leukocytesobtained from healthy human donors. The mononuclear cells were isolatedusing a Ficoll gradient, washed and incubated in RPMI supplemented with10% fetal calf serum (FCS) and antibiotics in petri dishes at 37° C.humidified CO₂ incubator. After 2 h, the non-adherent cells wereisolated and applied on nylon-wool columns (1.5 h). CD4⁺ T cells werethen negatively selected by exposure of the cells to a cocktail ofanti-CD8, CD19, and CD14 monoclonal antibodies (mAb) conjugated tomagnetic-beads (Advanced Magnetics, MA). Unbound cells were recoveredand their phenotype was examined. Purity of the CD4⁺ T cells was alwaysgreater than 92% as determined by FACScan.

The purified CD4⁺ T cells were radioactively labeled with ⁵¹ [Cr] (NewEngland Nuclear) in RPMI+20% FCS for 2 h and washed. The cells werecounted and seeded (0.2×10⁵ cells) on the precoated microtiter wells inthe presence or the absence of the various inhibitors. Coating was madeeither with FN, the 120 kD cell-attachment fragment of FN or withcontrol adhesive protein laminin (LN). The inhibitors were varioussurrogates according to the invention, other test molecules or mAb.After 30 min incubation (in 37° C. CO₂₋ humidified incubator) the Tcells were activated by 10 ng/ml phorbol myristate acetate (PMA) and thepercent of T cells attached to the protein substrates was measured.Unbound cells were washed away after 20-30 min, the bound cells werelysed and their radioactivity was measured using radioactive counter.The amount of the radioactivity of the cell lysates represent thematrix-adherent cells and percent binding was calculated in comparisonto the total radioactivity added to the wells. Activated T cell adhesionto control wells or to wells coated with BSA was always 2-5%; the levelof adhesion of the non-activated T cells was always below 5%. Whereindicated, 1/200 diluted mAb anti-CD29 (anti-β1 mAb Serotec, GB), or1/400 diluted anti-VLA5 (the β1α5 FN-integrin receptor) mAb (TeliosPharm. Inc. San Diego), or 0.2 mM of ARG-Gly-Asp (RGD; SEQ ID No:I),Gly-Arg-Gly-Asp-Ser-Pro-Lys (GRGDSPK; SEQ ID No:16) orGly-Arg-Gly-Glu-Ser-Pro (GRGESP; SEQ ID No:14) peptides (Sigma) wereused. The tested non-peptidic surrogates, 0.2 mM in PBS, were used topretreat the T cells for 15 min before seeding the cells *, P<0.05. Theresults shown in Tables 1A, 1B and 1C represent data obtained fromseveral experiments that produced essentially similar results.

In the first series of experiments shown in Table 1A, in which the 120kD fragment of FN was used, activation of the T cells resulted in celladhesion to both FN or LN (none). Blocking studies using various mAb tospecific integrin sites revealed that T cell binding to both proteins ismediated by β1-VLA integrins: anti-β1 mAb (anti-CD29 mAb) inhibited celladhesion to both proteins whereas anti-VLA-5 mAb inhibited T celladhesion to FN but not to LN. T cell adhesion to FN was specificallyinhibited by 0.2 mM RGD or GRGDSPK peptides but not by the controlpeptide GRGESP. The four RGD surrogates, AC-4. AC-14, SF-6,5 and AC-15inhibited T cell adhesion to FN but not to LN, with a most prominentinhibition exerted by the SF-6,5 surrogate. The ARG-Gly-Glu (RGE; SEQ IDNo:7) surrogate SF-6,6 and the amino compound SFN-70 did not inhibit Tcell adhesion to both FN and LN.

The inhibitory effect of the RGD analogues on T cell adhesion is not dueto a toxic effect since these compounds did not inhibit T cell adhesionto LN nor did they interfere with PMA or amitogen(phytohemagglutinin)-induced T cell proliferative responsesconducted for 48-72 h (data not shown). Thus, the non-peptidic RGDsurrogates specifically interfered with T cell adhesion to FN.

                  TABLE 1A                                                        ______________________________________                                        Specific inhibition of CD4.sup.+ T cell Adhesion                              to FN by RGD Surrogates                                                                  Adhesion of activated CD4.sup.+                                               T cell to: (% inhibition)                                          Inhibitor of T cell                                                                        120 kD                                                           adhesion     fragment of FN                                                                              LN                                                 ______________________________________                                        None         43 ± 4           55 ± 5                                    anti-CD29 mAb                                                                              10 ± 2                                                                             *     (83)  11 ± 2                                                                           *   (80)                               anti-VLA5 mAb                                                                              8 ± 2                                                                              *     (82)  52 ± 4 (0)                                RGD          38 ± 3     (12)  57 ± 3 (0)                                GRGDSPK      20 ± 2                                                                             *     (54)  53 ± 4 (0)                                GRGESP       46 ± 3     (0)   52 ± 3 (0)                                AC-4         16 ± 3                                                                             *     (47)  57 ± 4 (0)                                AC-14        25 ± 4                                                                             *     (42)  55 ± 6 (0)                                SF-6,5       22 ± 2                                                                             *     (49)  55 ± 6 (0)                                AC-15        35 ± 5                                                                             *     (19)  52 ± 4 (0)                                SF-6,6       46 ± 2     (0)   52 ± 6 (0)                                SFN-70       42 ± 5     (0)   49 ± 6 (0)                                ______________________________________                                    

In a second series of experiments, FN was used in the adhesion assaycarried out with the RGD surrogates NS-8, NS-11 and NS-15 in comparisonto ARG-Gly-Asp-Ser (RGDS; SEQ ID No:8) peptide. The results shown inTable 1B indicate that the compound NS-11 is a better inhibitor of celladhesion than the RGDS peptide.

                  TABLE 1B                                                        ______________________________________                                        Evaluation of inhibition of CD4.sup.+ T cell adhesion                         to FN by cyclic RGD surrogates                                                Inhibitor of T cell                                                                       % Adhesion of activated                                                                        % Inhibition                                     adhesion    CD4.sup.+ to FN  of adhesion                                      ______________________________________                                        None        65               --                                               RGDS        22               (67)                                             NS-11       19               (71)                                             NS-15       27               (59)                                             NS-8        45               (31)                                             ______________________________________                                    

In a third series of experiments, FN was used in the adhesion assay withthe surrogates SF-6,5 and NS-11 and compared to the peptideGly-Arg-Gly-Asp-Ser-Pro (GRGDSP; SEQ ID No:13). The results are shown inTable 1C.

                  TABLE 1C                                                        ______________________________________                                        Inhibition of CD4.sup.+ T cell adhesion to FN by RGD surrogates                                       % Adhesion of activated                               Inhibitor of            CD.sup.+ T cell to FN:                                T cell adhesion                                                                          Conc. (μg · ml)                                                                (% inhibition)                                        ______________________________________                                        GRGDSP     25           40                                                               50           50                                                               100          75                                                    SF-6,5     25           10                                                               50           25                                                               100          38                                                               200          55                                                    NS-11      25           30                                                               50           50                                                               100          85                                                    ______________________________________                                    

Example 16. Inhibition of tumor cell adhesion

To exert their metastatic activity, tumor cells must penetrate bloodvessel walls. Since RGD containing peptides have been shown to inhibitmetastasis in vivo, it was investigated whether the RGD surrogates ofthe invention inhibit tumor cell adhesion to the FN and vitronectin (VN)components of the ECM.

To examine the adhesive properties of tumor cells, 1 μg/50 μl/well of FNor 0.3 μg/well of VN were added to 96-flat bottom microtiter wells for12 h. Unbound proteins were then washed away and remaining binding siteswere blocked with 0.1% BSA added to the wells for 2 h and washed. MurineB16-melanoma F-1 cells were metabolically labeled with ³⁵ S-methionine(New England Nuclear) for 2 h, chased for 18 h and extensively washed.The cell suspension was resuspened in RPMI supplemented with 1% BSAcontaining 1 mM CaCl₂ and MgCl₂. Tumor cell adhesion to control wells orto wells coated with BSA was always 2-5%. The tested non-peptidicsurrogates, 0.2 mM in PBS, were used to pretreat the tumor cells for 15min before seeding the cells *, P<0.05. The results are shown in Tables2A and 2B.

In a first series of experiments, the potential inhibitory action of theRGD surrogate SF-6,5 on adhesion of B16 melanoma cells to FN or VN wascompared to that of the RGE surrogate SF-6,6 and to that of RGD, GRGDSPKand GRGESP peptides. The results are shown in Table 2A.

                  TABLE 2A                                                        ______________________________________                                        Inhibition of tumor cells adhesion to FN                                      and VN using RGD surrogates                                                                 % Adhesion of B16-melanoma cells                                Inhibitor of tumor cell                                                                     to: (% inhibition)                                              adhesion      FN           VN                                                 ______________________________________                                        None          75 ± 5          68 ± 5                                    RGD           70 ± 3     (7)  66 ± 6 (0)                                GRGDSPK       15 ± 4                                                                             *     (80) 10 ± 2                                                                           *   (86)                               GRGESP        73 ± 5     (0)  66 ± 8 (0)                                SF-6,5        34 ± 4                                                                             *     (55) 40 ± 5                                                                           *   (42)                               SF-6,6        75 ± 9     (0)  70 ± 7 (0)                                ______________________________________                                    

The B-16 murine melanoma cell adhesion to FN was found to be inhibitedby the GRGDSPK peptide, but not by RGD or the RGE peptides, nor by theRGE-surrogate SF-6,6. Nevertheless, the RGD surrogate SF-6,5 inhibitedtumor cell adhesion to both FN and VN.

In a second set of experiments carried out in vivo, we were able toclearly demonstrate an inhibition of tumor cell-induced metastases inC57BL/6 mice by i.v. daily administration of 25 μg of compound SF-6,5per mouse after the induction of metastasis. Both the native peptideGRGDSP and the compound SF-6,6 failed to inhibit metastases.

In a further set of experiments, the inhibitory activity of the RGDsurrogates SF-6,5 and NS-11 on the adhesion of B16 murine melanoma cellsto FN was compared to that of the GRGDSP peptide. The results are shownin Table 2B.

                  TABLE 2B                                                        ______________________________________                                        Inhibition of tumor cell adhesion to                                          FN by RGD surrogates                                                                                  % Adhesion of activated                               Inhibitor of tumor      tumor cells to FN:                                    cell adhesion                                                                             Conc. (μg · ml)                                                               (% inhibition)                                        ______________________________________                                        GRGDSP      25          30                                                                50          40                                                                100         65                                                    SF-6,5      25          10                                                                50          20                                                                100         30                                                                200         45                                                    NS-11       25          35                                                                50          45                                                                100         70                                                    ______________________________________                                    

Example 17. Inhibition of platelet aggregation

To investigate the inhibitory role of the RGD analogues on adhesion, theplatelet GPIIb-IIIa receptor which mediates platelet aggregation uponactivation was used as a model.

Platelet concentrates were prepared from human whole blood in 10%adenine-citrate-dextrose in Fenwall bags (Baxter Travenol, Israel)followed by standard AABB protocol. Platelet rich plasma was prepared bycentrifugation (2500 rpm for 5 min). Samples of platelets were countedin a Minos AST Cell Counter (Levoiselle, France). Cell aggregation wasinduced by 5 mM ADP and monitored at 695 mm in a 4-channel Aggregometer(Bio-Data, Hatboro, Pa.). To evaluate the effect of the various RGDpeptides and the peptide analogues, the platelet rich plasma waspre-incubated with the various inhibitors for 10 min at 37° C. with 10μl solutions followed by the induction of aggregation.

FIG. 3 shows a dose-dependent curve of inhibition of plateletaggregation by the compounds SF-6,5 (filled circles) and SF-6,6 (emptycircles). Compound SF-6,5, but not SF-6,6, was found to inhibit theaggregation of platelet rich plasma in a dose-dependent fashion withIC₅₀ of 0.3 mM.

To examine the specificity of the inhibitory effect of the RGD-analogueson platelet aggregation, the cells were treated with various peptidic(RGD and GRGDSP) and nonpeptidic (SF-6,6, SF-6,5 and AC-15) RGDanalogues used at a fixed sub-saturating concentration of 0.5 mM. FIG.4shows inhibition of platelet aggregation using 10 μg/ml concentration ofthe inhibitors (the results shown here summarizes the data obtained froma total of 4 experiments). These results show that the tripeptide RGDitself was not an effective inhibitor while the larger peptide GRGDSPKexerted a marked inhibitory effect on platelet aggregation. In addition,the inhibitory effect of the RGD surrogates SF-6,5 and AC-15 was evenhigher than that of the GRGDSPK peptide. The control surrogate, compoundSF-6,6, had a very limited inhibitory effect on platelet aggregationreflecting the inability of RGE to inhibit platelet aggregation.

Example 18. Inhibition of platelet aggregation by NS-11

An in vitro assay as described in Example 17 was carried out with thecompound NS-11 to examine its ability to interfere with plateletaggregation, and compared it to the SF-6,5 surrogate.

Table 3 summarizes the results obtained in analyzing the effect of bothmolecules on the ADP-induced platelet aggregation.

                  TABLE 3                                                         ______________________________________                                        NS-11 mediated inhibition of platelet aggregation                                                     Percent inhibition                                    Compound    Concentration                                                                             of aggregation                                        ______________________________________                                        GRGDSP      0.1 mM      75                                                    SF-6,5      0.1 mM      25                                                                0.3 mM      55                                                    NS-11       0.1 mM      90                                                                0.3 mM      100                                                   ______________________________________                                    

It can be summarized that the three compounds tested were found toinduce inhibition of platelet aggregation. The surrogate SF-6,5 had amild, though significant effect on aggregation: comparing its effect tothat of the RGD-containing peptide on a mM basis, reveals that thismolecule had slightly lower effect on the cell function. The resultsobtained indicate that NS-11 is a better inhibitor of plateletaggregation than SF-6,5. Moreover, this molecule is a significantlybetter inhibitor than the RGD-containing peptide. Its effect at theconcentration of 0.1 mM is better than that of the RGD-peptide, used atthe same concentration, by almost 20%.

Example 19. Inhibition of platelet aggregation by NS-8, NS-11, and NS-15

Platelet rich plasma (PRP) was prepared from acid/citrate/dextroseanti-coagulated fresh human blood by differential centrifugation.Platelet aggregation was induced by 5 mM ADP and monitored at 695 nm bya 4-channel Aggregometer (Bio-Data, PA). To evaluate the effect of theRGDS peptide and the. NS-8, NS-11 and NS-15 analogs, the PRP waspreincubated with the inhibitors for 10 minutes at 37° C., prior to theinduction of aggregation. As seen in FIG. 5, compound NS-11 was a betterinhibitor of platelet aggregation than either NS-8 and NS-15. Moreover,compound NS-11 was found to inhibit platelet aggregation better than theRGD-containing peptide (RGDS). In fact, 50% inhibition of plateletaggregation was achieved using a 30-fold lower concentration of NS-11than that of RGDS.

Example 20. Inhibition of the binding capacity of anti-GPIIb-IIIa mAb(PAC-1) to platelets

To investigate whether the RGD surrogates actually bind to theGPIIb-IIIa integrin, their ability to compete with a monoclonal antibody(mAb) specific to GPIIb-IIIa was investigated. This mAb, designatedPAC-1, specific for the activated receptor, binds GPIIb-IIIa in anRGD-dependent manner (Taub, R. et al. (1989) J.Biol.Chem. 264: 259).

ADP-activated platelets were incubated with FITC-conjugated PAC-1 mAb inthe presence of various peptidic and non-peptidic compounds, as follows:the platelet rich plasma were gel-filtrated into modified Tryode'ssolution (137.5 mM Nacl, 4 mN Hepes, 2.6 mM KCl, 1 mM MgCl₂, 3.3 mM NaH₂PO₄, 5.6 mM glucose at pH 7.4) containing 350 μg/ml BSA (which was usedas the incubation buffer in further steps; Tryode's/BSA). The finalcount in the cell assays was 2×10⁶ per ml. The cells were then activatedwith 10 μM ADP and epinephrine. To examine RGD peptides and relatedanalogues as competitors for the binding capacity of the PAC-1 mAb toplatelets, the cells were incubated in 50 μl Tryode's/BSA supplementedwith 1 mM CaCl₂ for 30 min in 25° C., in the presence of 0-500 μMpeptides or peptide-analogues with 10 μg/ml FITC labeled PAC-1. Thefluorescence profile of the cells was determined using FACScan (BecktonDickenson) at 488 nm. In FIG. 6: GRGDSPK (empty circles), GRGESP (filledtriangles), compound SF-6,5 (empty triangles), compound SF-6,6 (filledcircles) (the data shown here represent results obtained in one of threeexperiments which were essentially identical).

The results shown in FIG. 6 indicate that both the RGE peptide and theRGE analogue compound SF-6,6 failed to inhibit PAC-1 binding to theplatelet integrin receptor. However, the GRGDSPK and the RGD surrogate,compound SF-6,5, inhibited PAC-1 staining of the cells in adose-dependent manner. Thus, the ability of the RGD surrogate compoundto inhibit platelet aggregation could be attributed to directinterference with the RGD binding site on the GPIIb-IIIa receptor.

Example 21. Inhibition of DTH response to OX by treatment of mice withRGD surrogate

To examine the regulatory role of SF-6,5 on T cell immunity andlymphocyte migration in vivo, a delayed-type hypersensitivity (DTH)reaction experiment was performed in which groups of BALB/c mice (6 miceper group) were sensitized on the shaved abdomen with the skin allergen4-ethoxymethylene-2-phenyl oxazolone (OX) (10 μl of 3% OX inacetone/olive oil) and challenged again 5 days later by applying OX totheir ears. The increment in ear swelling was recorded 24 hours later asa measure of DTH. The individual measuring of ear swelling was blindedto the identity of the groups of mice. GRGDS, RGD surrogate compoundSF-6,5 and RGE surrogate compound SF-6,6 were administrated I.V. in 200μl PBS into the tail vein on the indicated days. Control groups of micewere treated identically with PBS.

The results shown in Table 4 indicate that treatment with compoundSF-6,5 but not with SF-6,6, inhibited the DTH response best when themice were injected for 6 days (groups 7 and 8, respectively). Inaddition, the RGD surrogate was found to be a better inhibitor of theDTH response than the GRGDS peptide, most probably due to shorterphysiological retention-times of the latter (group 3). Indeed, as shownin Example 14, it was found that the compound SF-6,5, unlike the GRGDSpeptide, was completely resistant to trypsin-induced hydrolysis. Theresults obtained in these groups did not differ significantly from thoseobtained in the positive control group (data not shown),*: P<0.01; Pvalues were measured in relation to group 2, the positive control group.These findings indicate that modulation of cell-mediated immunereactions in vivo may be achieved by relatively low doses ofnon-peptidic RGD analogue, most probably by means of interfering withlymphocyte migration.

                  TABLE 4                                                         ______________________________________                                        Inhibition of DTH response to OX by treatment                                 of mice with RGD surrogate                                                                    Elicitation of OX-mediated                                                    DTH response                                                                            Δ Ear                                         Treatment of mice OX-     swelling                                                             injected sensiti-                                                                            (×10.sup.-2                                                                     %                                     Group Compound:  on days: zation                                                                              mm ± SD)                                                                           inhibition                            ______________________________________                                        1     None       --       No    2 ± 2  --                                  2     None       --       Yes   21 ± 2 --                                  3     GRGDSPK    1 to 6   Yes   17 ± 3 20                                  4     SF-6,5     1        Yes   16 ± 3 20                                  5                1, 3     Yes   13 ± 2 34                                  6                1, 3, 5  Yes   8 ± 2                                                                            *   62                                  7                1 to 6   Yes   2 ± 1                                                                            *   95                                  8     SF-6,6     1 to 6   Yes   23 ± 4 None                                ______________________________________                                    

REFERENCES

Adler, M., Lazarus, A., Dennis, M. S. & Wagner, G. (1991) Science253:445-447.

Alon, R. et al., (1990) BBRC 170:1236-1241.

Barany, N. & Merrifield, R. B. The Peptides. Analysis, Synthesis andBiology (Gross, E. Ed.) Academic Press, New York, 1980, p. 2.

D'Souza, S. E., Ginsberg. M. H., Matsueda, G. R. & Plow, E. F. (1991a)Nature 350: 66-68.

D'Souza, S. E. et al. (1991b) TIBS 16:246.

Elices, M. J., Urry, A. & Hemler, M. E. (1991) J. Cell Biol.112:169-181.

Humphries, M. J., Olden, K. & Yamada, K. M. (1986) Science 233:467-470.

Hynes, R. O. (1990) Fibronectins, Sperger, Verlag.

Hynes, R. O. (1992) Cell 69:11-25.

Mazerolles, F. et al. (1988) Cell 55:497-504.

Mazerolles, F., Amblard, F., Lumbroso, C., Lecomte, O., Van de Moortele,P. F., Barbat, C., Piatier-Tonneau, D. & Fisher, A. (1990) Eur. J.Immunol. 20:637-644.

Mould, A. P. et al. (1991) J. Biol. Chem. 266:3579-3585.

Ouaissi, M. A., Cornette, J., Afchain, D., Capron, A., Gras-Masse, H.and Tartar, A. (1986) Science 234:603-607.

Philips, D. R., Charo, I. F. & Scarborough, R. M. (1991) Cell65:359-362.

Ruoslahti, E. (1988) Annu. Rev. Biochem. 57:375-391.

Ruoslahti, E. & Giancotti, F. G. (1989) Cancer Cell 1:119-126.

Ruoslahti, E. et al. (1989) Cancer Cells 111:249.

Ruoslahti, E. (1991) J. Clin. Invest. 87:1-5.

Shimizu, Y., van Seventer, G. A., Horgan, K. J. & Shaw, S. (1990) Nature345:250-253.

Shimizu, Y. & Shaw, S. (1991) FASEB J. 5:2292-2299.

Springer, T. A. (1990) Nature 346:425-434.

Taub, R., Gould, R. J., Garsky, V. M., Ciccarone, T. M., Hoxie, J.,Friedman, P. A. & Shattill, S. J. (1989) J. Biol. Chem. 264:259-265.

Van Seventer, G. A. et al. (1991) Curr. Opin. Immunol. 3:294.

Vogel, B. E. et al (1992) J. Cellular Biochem. 16F:154.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgGlyAsp                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgAlaAsp                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgGluAsp                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgPheAsp                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgTyrAsp                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                      AspGlyArg                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ArgGlyGlu                                                                      1                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ArgGlyAspSer                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ArgAlaAspSer                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                      (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ArgPheAspSer                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgTyrAspSer                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   ( xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                     SerAspGlyArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GlyArgGlyAsp SerPro                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyArgGlyGluSerPro                                                             15                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyArgGlyAspSer                                                               1 5                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyArgGlyAspSerProLys                                                         1 5                                                                       

We claim:
 1. A method of inhibition of cell adhesion dependent oncell-adhesion-motif ARG-GLY-ASP (RGD; SEQ ID No:1) interactions whichcomprises administering a compound of the formula I

    H.sub.2 N--C(═NH)--NH--CH.sub.2 --A--CH.sub.2 --CO.sub.2 H (I)

and pharmaceutically acceptable salts thereof, wherein A is a chain of 9atoms selected from the group consisting of: ##STR7## wherein each of x,n and m is at least 1; in chains (i) and (ii) n is at most 6; in chains(iii) and (iv) the sum of x+n+m is 5; and the sum of n+m is 4 in chain(v) and 3 in chain (vi).
 2. The method according to claim 1, wherein thecompound has the formula ##STR8## wherein n is at least 1 and at most 8.3. The method according to claim 1, wherein the compound has the formula##STR9## wherein n is at least 1 and at most
 8. 4. The method accordingto claim 1, wherein the compound has the formula ##STR10## wherein eachof x, n and m is at least 1 and the sum of x+n+m is
 7. 5. The methodaccording to claim 1, wherein the compound has the formula ##STR11##wherein each of x, n and m is at least 1 and the sum of x+m+n is
 7. 6.The method according to claim 1, wherein the compound has the formula##STR12##
 7. The method according to claim 1, wherein the compound hasthe formula ##STR13##
 8. The method according to claim 1, wherein thecompound has the formula ##STR14##
 9. The method according to claim 1,wherein the compound has the formula ##STR15##
 10. The method accordingto claim 1, wherein the compound has the formula ##STR16##
 11. Themethod according to claim 1, wherein the compound has the formula##STR17##